1. Field of the Invention
This invention relates to a novel genomic DNA sequence (MEFV) encoding a protein (pyrin) associated with familial Mediterranean fever (FMF). More specifically, the invention relates to the isolation and characterization of MEFV, and the correlation of mutations in MEFV with FMF disease.
2. Background of the Invention
Familial Mediterranean Fever (FMF) is a recessively inherited disorder characterized by dramatic episodes of fever, serosal inflammation and abdominal pain. This inflammatory disorder is episodic, with self-limited bouts of fever accompanied by unexplained arthritis, sterile peritonitis, pleurisy and/or skin rash. Patients often develop progressive systemic amyloidosis from the deposition of the acute phase reactant serum amyloid A (SAA). In some patients, progressive systemic amyloidosis can lead to kidney failure and death. The factors which incite an episode are unclear.
FMF is observed primarily in individuals of non-Ashkenazi Jewish, Armenian, Arab and Turkish background. Although rare in the United States, incidence of FMF in Middle Eastern populations can be as high as 1:7 in Armenian populations and 1:5 in non-Ashkenazi Jewish populations.
FMF attacks are characterized by a massive influx of polymorphonuclear leukocytes (PMNs) into the affected anatomic compartment. At the biochemical level, patients have been reported to have abnormal levels of C5a inhibitor (Matzner and Brzezinski, xe2x80x9cC5a-inhibitor deficiency in peritoneal fluids from patients with familial Mediterranean fever,xe2x80x9d N. Engl. J. Med., 311:287-290 (1984)), neutrophil-stimulatory dihydroxy fatty acids (Aisen et al, xe2x80x9cCirculating hydroxy fatty acids in familial Mediterranean fever,xe2x80x9d Proc. Natl. Acad. Sci. USA, 2:1232-1236 (1985)), and dopamine xcex2-hydroxylase (Barakat et al, xe2x80x9cPlasma dopamine beta-hyroxylase: rapid diagnostic test for recurrent hereditary polyserositis,xe2x80x9d Lancet, 2:1280-1283 (1988)). Although linkage studies have placed the gene causing FMF (designated MEFV) on chromosome 16p (Pras et al., xe2x80x9cMapping of a gene causing familial Mediterranean fever to the short arm of chromosome 16,xe2x80x9d N. Engl. J. Med., 326:1509-1513 (1992); Shohat et al., xe2x80x9cThe gene for familial Mediterranean fever in both Armenians and non-Ashkenazi Jews is linked to the xcex1-globin complex on 16p: evidence for locus homogeneity,xe2x80x9d Am. J. Hum. Genet., 51:1349-1354 (1992); Pras et al, xe2x80x9cThe gene causing familial Mediterranean fever maps to the short arm of chromosome 16 in Druze and Moslem Arab families,xe2x80x9d Hum. Genet., 94:576-577(1994); French FMF Consortium, xe2x80x9cLocalization of the familial Mediterranean fever gene (FMF) to a 250 kb-interval in non-Ashkenazi Jewish founder haplotypes,xe2x80x9d Am. J. Hum. Genet., 59:603-612(1996)), the genetic basis of FMF has not previously been identified.
Current treatment regimens for FMF include daily oral administration of colchicine. Although colchicine has been shown to cause near complete remission in about 75% of FMF patients and prevent amyloidosis, colchicine is not effective in all patients. Therefore, there is a need for new treatments for colchicine-resistant patients.
Additionally, there is a need for an accurate diagnostic test for FMF. Patients having FMF in countries where the disease is less prevalent often experience years of attacks and several exploratory surgeries before the correct diagnosis is made.
The invention provides a novel genomic nucleic acid sequence (MEFV) (SEQ ID NO:1), shown in FIG. 1, encoding the protein pyrin which is associated with familial Mediterranean fever (FMF). The corresponding cDNA sequence (v75-1) (SEQ ID NO:2) and encoded amino acid sequence (SEQ ID NO:3) are shown in FIG. 2. The invention is also directed towards fragments of the DNA sequence that are useful, for example, as hybridization probes for diagnostic assays or oligonucleotides for PCR priming. Additionally, the invention is directed towards the corresponding sequence for the RNA transcript and fragments thereof.
Another aspect of the invention provides the amino acid sequence for a protein associated with FMF. This protein is called pyrin, to connote its relationship to fever. The invention is directed towards both the full length amino acid sequence, fusion proteins containing the amino acid sequence and fragments thereof. These proteins are useful, for example, as antigens to produce specific anti-pyrin antibodies to be used as agents in diagnostic assays. Alternatively, the protein may be used in therapeutic compositions.
Mutations in pyrin result in FMF. Therefore, the invention is also directed towards mutants of the nucleic acid and amino acid sequences associated with FMF. In particular, the invention discloses three missense mutations, clustered in within about 40 to 50 amino acids, in the highly conserved rfp (B30.2) domain (SEQ ID NO:5) at the C-terminal of the protein. These mutants include M680I, M694V, K695R and V726A, each of which is associated with FMF.
Additionally, the invention includes methods for diagnosing a patient at risk for having FMF using the nucleic acid and/or amino acid sequences of the invention. Such methods include, for example, hybridization techniques using nucleic acid sequences, PCR-amplification of MEFV, and immunoassays using anti-pyrin antibodies to identify mutations is MEFV or pyrin which are indicative of FMF.